Earlier we showed that altering the expression of wild type Hsp40s (J-proteins), nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain proteins can either enhance or impair propagation of different prions in both wild type and Hsp70 mutant yeast (S. cerevisiae) cells. We also identified several mutant alleles of Hsp40s and TPR proteins that impair or enhance propagation of different prions. Our further work pointed to the fact that many, if not all, of the observed effects that these co-chaperones have on prions are mediated by the various ways they can regulate activities of Hsp70. Yeast and mammals have a similar number of cytosolic Hsp70s, and we showed mammalian Hsp70s can provide the essential Hsp70 functions required to support viability of yeast. However, while yeast has about twenty J-protein co-chaperones, the human genome encodes roughly fifty J-proteins. These observations suggest that diversification of intrinsic Hsp70 activities has diminished and that the ability of Hsp70 to perform additional or more complex tasks in mammals has expanded through the amplification of J-protein (and other) Hsp70 co-chaperones that regulate, recruit and fine-tune its activities. The extent to which various J-proteins provide specificity to Hsp70 function ion vivo by providing their own unique substrate interactions or by their ability to recruit and regulate basic Hsp70 activities is far from understood. By replacing the yeast Hsp70-dependent disaggregation machinery with homologous bacterial components, we showed that this machinery requires species-specific interactions of all components except for the J-proteins, which functioned across species. Exploiting this promiscuity, we showed the major Hsp40 class yeast J-proteins, Ydj1 and Sis1, define and specify separate functions of this machinery required for protection from heat stress and prion propagation, respectively. We went on to show these J-proteins similarly specify functions of the endogenous yeast disaggregation machinery. Additionally we showed that the substrate-binding regions of the J-proteins determine the functional specificity not only of the disaggregation machinery, but also Ydj1-dependent functions of the Hsp90 machinery. These findings point to substrate discrimination as a major factor in how Hsp40 type J-proteins specify Hsp70 function. Altering abundance or function of Hsp70 and its co-chaperones, in particular Hsp40s, reduces pathology in several models of amyloid and other protein folding disorders. Our work provides insight into functions of this chaperone system that can help guide strategies for using chaperones as targets for therapy in such diseases.